The p53 gene product is an ideal target for enhancement of the cellular immune response to cancer. Almost 50 percent of all cancers have p53 mutations. p53 overexpression is an independent predictor of more aggressive cancer and cancer related mortality. What makes p53 such an attractive target for an adaptive immune response is that the intracellular concentration of non-mutated p53 is normally very low, and cells expressing wild type p53 at low levels will likely escape autoimmune attack by an enhanced immune response to over-expressed mutant p53. Results from our laboratory demonstrate that in preclinical models it is possible to reject established p53-overexpressing tumors without stimulating autoimmunity by immunization with an attenuated poxvirus referred to as modified vaccinia Ankara expressing p53(MVAp53). MVA has several advantages as a vaccine delivery vector including avirulence, low inflammatory response and minimal pathogenicity in vertebrates including humans. We also determined that the therapeutic efficacy of MVAp53 is enhanced by the coadministration of synthetic ODN containing unmethylated CpG (cytosine-phosphateguanine) motifs (CpG ODN). We proposed a Phase I dose-escalation study of MVAp53 designed to evaluate the safety and immunogenicity of MVAp53 administered alone and with CpG ODN adjuvant. The trial will be conducted in patients with advanced GI malignancies which overexpress p53. Specific aim I tests the hypothesis that MVAp53 administered alone or with CpG ODN adjuvant will be well tolerated and associated with limited toxicity. Consented research subjects will receive up to 4 doses of vaccine, and a dose escalation will be conducted to determine if therapeutically relevant doses can be safely administered. Specific aim II tests the hypothesis that immunization of GI cancer patients with MVAp53 will result in enhanced cellular and humoral immune responses to p53 and that p53 specific immune responses will be enhanced through the coadministration of CpG ODN. Immunogenicity will be assessed using an ELISA assay for humoral response, lymphoproliferation for CD4+ T cell response and intracytoplasmic cytokine assays and IFN-gamma and IL-4 by ELISPOT assays for assessment of cellular immune response. For each assay, the pre-vaccine values will be compared to post-vaccine values. If differences exist between pre-vaccine and post-vaccine values, a determination will be made as to the vaccine regimen required to achieve p53 specific responses in the greatest proportion of patients. Successful demonstration of the stimulation of p53 specific immunity using this cell free vaccine approach will support the future large scale evaluation of p53 specific immunization alone or as a component of a multivalent vaccine approach and establish a new paradigm for cell free tumor vaccines targeting defined antigen.